Project Summary: Melanoma incidence continues to grow 3-7% per year while the incidence of most cancer is decreasing. Thus, melanoma etiology research remains a priority for disease control and treatment. Similarity of gene expression and pathological features between the Xiphophorus (platyfish) melanoma model and human melanoma allowed researchers to adapt discoveries from Xiphophorus model system to the human condition. The interspecies hybrid spontaneous Xiphophorus melanoma model results from Mendelian segregation of a mutant egfr gene, termed xmrk, and a hypothetical tumor suppressor termed R(Diff). R(Diff) is hypothesized to suppresses the activity of xmrk in healthy X. maculatus parental species, and in X. maculatus and X. hellerii interspecies hybrids. Considering the mutant egfr nature of the xmrk, R(Diff) represent an endogenous egfr regulator of unknown mechanism. Therefore, identifying the molecular genetic features whereby R(Diff) suppresses the deleterious activity of a mutant egfr is of significant value, not only to melanoma treatment, but also development of new treatment strategies for EGFR related cancers. Our recent study has defined the R(Diff) tumor regulatory locus to a 5.8 Mbp locus on chromosome 5. Considering the large size and number of gene models (164 genes) encoded in this region, functional and mechanistic studies are impractical. Therefore, this proposal is designed to improve our understanding of the R(Diff) locus by examining the following aims: Aim 1: We will genotype expressed genes in Xiphophorus benign pigment cell lesions and spontaneous melanoma tumors, and define candidate R(Diff) loci on newly re-sequenced and gap-closed Xiphophorus genome assemblies. Aim 2: We will reduce the size of the candidate R(Diff) locus on chromosome 5 by genotyping R(Diff) candidates in large numbers of BC1 hybrid progeny. Aim 3: We will evaluate the effectiveness of R(Diff) candidate gene(s) in the ability to regulate the xmrk oncogene in the Xiphophorus pigment lesion cells and the xmrk-Tg medaka model.